Monoclonal antibody against CK8 protein and cell strain, preparation method and application thereof

A monoclonal antibody and hybridoma cell line technology, applied in the field of biomedical engineering, can solve cryptogenic liver cirrhosis and other problems, and achieve highly specific effects

Active Publication Date: 2021-06-11
FUZHOU MAIXIN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations in this gene cause cryptogenic liver cirrhosis

Method used

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  • Monoclonal antibody against CK8 protein and cell strain, preparation method and application thereof
  • Monoclonal antibody against CK8 protein and cell strain, preparation method and application thereof
  • Monoclonal antibody against CK8 protein and cell strain, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Polypeptide Synthesis and Chemical Coupling with Carrier Protein

[0020] According to the sequence and secondary structure analysis of the protein sequence with the accession number P05787 in Uniprot, the molecular weight of the CK8 protein with a total length of 483 amino acids is about 53 kDa. According to the predicted protein secondary structure (secondary structure) and surface accessibility (Surface Accessibility) parameters through the online server http: / / www.cbs.dtu.dk / services / NetSurfP / , and through its antigenicity index Analyzing the results, the amino acid sequence at position 340-365 was selected as the antigenic peptide for chemical synthesis (its sequence is shown in SEQID1 in the sequence listing), and for the convenience of coupling, a cysteine ​​was added to the carboxyl terminus of the antigenic peptide for Provides for sulfhydryl coupling. The coupling steps are as follows:

[0021] Select the maleamide-activated keyhole limpet hemocyan...

Embodiment 2

[0023] The establishment of embodiment 2 hybridoma cell lines

[0024] 1. Immunity

[0025] The cross-linked polypeptide in Example 1 was emulsified with complete Freund's adjuvant (Sigma, F5881), immunized 3 ICR mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and each mouse was injected subcutaneously in the abdomen. Rats at 6 o'clock, the dose is 20μg / only. Immunization was boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20 μg per mouse. 7 days after the third booster immunization, indirect ELISA (wavelength 450nm) was used to detect the polyantibody titer of the anti-immunogen in the mouse serum. 20μg / only.

[0026] 2. Cell Fusion

[0027] Aseptically prepare the mouse splenocyte suspension that reaches the immune standard, mix it with mouse myeloma cell sp2 / 0 (ATCCNumberCRL-8287) at a ratio of 5:1, and centrifuge at 1500rpm for 5min. After the supernatant was disc...

Embodiment 3

[0030] Example 3 Preparation of monoclonal antibody by ascites induction method

[0031] 1. Ascites preparation

[0032] The cells in the logarithmic growth phase were washed with serum-free medium and suspended, counted about 5×10 5, 1ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4000rpm for 10min at 4°C. Carefully suck out the ascites in the middle and collect in a centrifuge tube, and store at 4°C or -20°C.

[0033] 2. Purification of monoclonal antibodies

[0034] Antibody was purified from ascitic fluid by HiTrap rProtein A FF (GE Company) affinity chromatography according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. Purified antibodies were stored at -20°C.

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Abstract

The invention relates to a monoclonal antibody capable of recognizing a human leukocyte differentiation antigen CK8, a secretory cell strain, a preparation method thereof and application in immunodetection. According to the technical scheme, 340-365 amino acid sequences at the C tail end of CK8 protein are selected as antigen peptides, and the antigen peptides are coupled with KLH carrier protein to obtain the immunogen; the amino acid sequence of the antigen peptide is shown as SEQID1, a mouse is immunized, and the mouse hybridoma cell strain 14C2 capable of efficiently secreting the anti-CK8 protein monoclonal antibody and the anti-CK8 protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the CK8 protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

technical field [0001] The invention relates to the field of biomedical engineering, in particular to an anti-CK8 protein monoclonal antibody and its cell line, preparation method and application. Background technique [0002] Cytokeratin 8 (CK8), one of the important structures of the cytoskeleton, is located on human chromosome 12q13 and is expressed in simple epithelium, tubular epithelium and pseudostratified epithelium. CK8 / 18 is the main component of intermediate fibers in monolayer epithelial cells, mainly in the form of dimers; it is distributed in cancer-prone sites such as gastrointestinal tract, liver, pancreas and breast. CK8 is specifically expressed in single-layer epithelial tissue, localized in the cytoplasm, and consists of 483 amino acids. Cytokeratin 8 has three functional domains, including a centrally located, conserved a-helix-forming rod-shaped domain, and an NH2-terminal head domain and a COOH-terminal tail domain connected to it. The rod-shaped dom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C07K14/47C12N5/20G01N33/577G01N33/68G01N33/574
CPCC07K16/18C07K14/4741G01N33/577G01N33/68G01N33/57484G01N33/574C07K2317/56C07K2317/92G01N2333/4742
Inventor 蒋宁城杨清海陈惠玲王小亚傅椿辉林婷
Owner FUZHOU MAIXIN BIOTECH CO LTD
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